Virus purification and formulation process

ABSTRACT

Disclosed herein is provided a virus purification and formulation process for purifying a flavivirus represented by one of a Yellow Fever Virus, Japanese Encephalitis virus, Dengue virus, and West Nile virus. The highly purified flavivirus virus product is characterized as having a low level of sucrose without significant virus loss such as that which is typically encountered by prior art virus purification processes. The disclosed process captures and purifies the virus, separating it from the host cell proteins and DNA, and leaving the host cell proteins and DNA behind. The process also can be used to inactivate and/or concentrate the virus sufficiently for use in formulations.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/163,288, filed on May 24, 2016, which is a continuation of U.S.patent application Ser. No. 14/423,394, filed on Feb. 23, 2015, whichclaims the benefit of PCT Patent Application No. PCT/US2013/55302, filedon Aug. 16, 2013, which claims benefit to U.S. Provisional ApplicationNo. 61/692,956, filed on Aug. 24, 2012, which are hereby incorporated byreference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to a method of purifyingbiologics, such as a virus or modified virus, and a method offormulating the biologic, for example, a virus and adjuvant.

BACKGROUND OF THE INVENTION

In order to purify a virus it is necessary to remove host cell proteinsand DNA from the virus sample. Unfortunately, current methods ofremoving host cell proteins and DNA are accompanied by loss of asignificant amount of virus. Typically, for a flavivirus, recovery ofthe virus from filtration processes are generally about ten to twentypercent and typically less than 10 percent. Following purification,another problem is aggregation or clumping together of the purifiedvirus particles.

Prior art methods of purification and concentration of a virus, e.g.,often use ultracentrifugation wherein sucrose is required in order torun the gradient for the separation. Such ultracentrifugation generallyresults in the undesirable presence of sucrose in the final virussample. In order to obtain a virus sample that is free of sucrose,tangential flow filtration (TFF) is frequently used to separate thevirus from proteins and other compounds. However, use of TFF for such apurification tends to result in significant loss of virus.

Prior art methods of purification and concentration of a virus, e.g.,often use ultracentrifugation wherein sucrose is required in order torun the gradient for the separation. Such ultracentrifugation generallyresults in the undesirable presence of sucrose in the final virussample. In order to obtain a virus sample that is free of sucrose,tangential flow filtration (TFF) is frequently used to separate thevirus from proteins and other compounds. However, use of TFF for such apurification tends to result in significant loss of virus. As well,currently used methods of virus purification thus have been accompaniedby ongoing problems including low yield or loss of virus, host cellprotein levels higher than desirable, high sucrose levels, andaggregation of the purified virus particles. Such methods have also beendifficult to use in single use or disposable technologies.

BRIEF SUMMARY OF THE DISCLOSURE

In accordance with the present invention there is provided a viruspurification process for producing highly purified virus product havingno residual sucrose without significant virus loss such as that which istypically encountered by prior art virus purification processes. Thedisclosed process captures and purifies the virus, separating it fromthe host cell proteins and DNA, and leaving the host cell proteins andDNA behind. The process also can be used to inactivate and/orconcentrate the virus sufficiently for use in formulations.

Also disclosed herein is a one-step process for producing a formulationwherein the concentrated virus particles are not aggregated or clumpedtogether. The disclosed process utilizes adjuvants and buffer exchangesto process the virus, and results in a final, buffered virusformulation. An embodiment of the process can also be used forformulation of other biologics.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described hereinafter withreference to the accompanying drawings, in which:

FIG. 1 is a process chromatogram for Experiment Y1626A, an embodiment ofthe new downstream process disclosed herein that is used to purify andformulate a harvest of modified YF virus.

FIG. 2 is a process chromatogram for Experiment Y1632A, an embodiment ofthe new downstream process disclosed herein that is used to purify andformulate a harvest of modified YF virus, comparing elutions usingCELLUFINE® sulphate and CAPTO™ Core 700.

FIG. 3 is a process chromatogram for Experiment Y1637A.

FIG. 4 is a process chromatogram for Experiment Y1639A using CELLUFINE®Sulfate column.

FIG. 5 is a process chromatogram for Experiment Y1639A using CAPTO™DeVirS Column.

FIG. 6 is a flow diagram for an embodiment of the old downstreampurification process and a flow diagram for an embodiment of the newdownstream purification process.

FIG. 7 is a flow diagram for the embodiment of the old downstreampurification process shown in FIG. 6 and a flow diagram for anembodiment of the new and improved downstream purification process.

FIG. 8 is a flow diagram for another embodiment of an old downstreampurification process and a flow diagram for an embodiment of the new andimproved downstream purification process, Number 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The inventors of the present subject matter have now discovered aprocess for preparing a highly purified biological composition, such as,for example, a virus. The process is particularly useful for purifying aflavivirus, e.g., a Yellow Fever Virus, Japanese Encephalitis virus,Dengue virus, and West Nile virus. The examples provided herein are forpurifying a Yellow Fever virus, but with no more than routineexperimentation, could be used to purify other types of viruses.

The disclosed process significantly reduces the loss of viral particlesas compared to prior art methods for obtaining a highly purified sampleof a biologic. Use of an embodiment of the disclosed method alsosignificantly lowers the amount of sucrose in the final purified productas compared to prior art methods of purification.

We now describe the development of a chromatographic process resultingin vaccine material with DNA and host cell contaminant levels at orbelow the level of detection while retaining high levels of virusrecovery. Disclosed herein is a Yellow Fever (YF) vaccine downstreampurification process designed to inactivate live YF virus, fragment VeroDNA, and remove any process contaminants, such as residual Vero hostcell proteins and DNA, residual BENZONASE® and beta(ß)-Propiolactone(BPL).

YF virus may be grown on Vero cells, harvested, inactivated, andpurified. Typically, prior art methods for downstream purification of avirus provide a YF virus recovery of only about 20 percent (%); and thefinal virus product for dosing includes a relatively high amount of HostCell Protein (HCP). A typical YF vaccine dose for a phase 1 clinicaltrial may be about 0.5 milliliters (ml) of YF virus in a suspension. Theremaining HCP in a dose purified by prior art methods may typically beabout forty-five thousand nanograms (45,000 ng/dose) wherein the dose isabout 8.3 log 10 Viral Equivalents (VE). VE is the Elisa unit for YF.

In contrast, the disclosed method provides, for the modified YF viruspurification tests run to date, a downstream virus recovery of fromabout 30 percent to about 100 percent; and for a dose of about 0.5milliliters (ml) of YF virus, a HCP level that is below the limit ofdetection of the commercially used vero cell HCP assay.

To purify the final virus product by reducing host cell proteins whilemaximizing virus recovery, we designed and executed a number ofexperiments.

Modified Yellow fever virus was prepared from the attenuated YF 17Dvirus available commercially as the vaccine, YF-VAX® (Sanofi Pasteur,Swiftwater Pa.). The attenuated YF 17D virus was adapted by serialpassage to replicate more efficiently in Vero cells derived from the WHOVero 10-87 Cell Bank and passaged in serum free medium. 10 serialpassages were used to modify the nucleotide sequence of the viral genomevirus to develop a seed virus with enhanced growth in Vero cells forpreparation of an inactivated Yellow Fever virus candidate. SeePCT/US2010/043010, “High Yield Yellow Fever Virus Strain With IncreasedPropagation In Cells”, filed 23 Jul. 2010 and published 3 Feb. 2011 asWO 2011/014416, and PCT/US2011/022347, filed 25 Jan. 2011. The entireteachings of the referenced PCT applications are incorporated herein byreference.

The master and working virus seeds were manufactured from theconditioned cell culture medium harvested from stationary cultures ofVero cells prepared from the Manufacturers' Working Cell bank (MWCB).For vaccine production, the working virus seed was used to infect Verocells prepared from the MWCB grown on CYTODEX™ 1 microcarriers in eithera 50 liter, single use bioreactor (working volume 25 to 40 L) or a 10 Lglass bioreactor (working volume 8 L) (Xcellerex, Inc., Marlborough,Mass.). The virus released into the cell culture medium was harvestedfrom about 5 to about 7 days after infection. From about 2 days to about3 days before harvest of the virus, the culture was re-fed with freshmedium. This re-feeding step has been shown to increase virus yield. SeePCT/US2010/043013, “Drain Down and Re-Feed of Microcarrier Bioreactor,”filed 23 Jul. 2010 and published 27 Jan. 2011 as WO 2011/011660, theentire teachings of which are incorporated herein by reference.

Some of our initial experiments in developing a purification process forthe virus are schematically represented in the flow chart on the leftside of each of FIG. 6 and FIG. 7, “Downstream Old Process.” One set ofsteps was: Harvest Clarification and BENZONASE® Addition; UltraFiltration and DiaFiltration (UF/DF); BPL Inactivation; CELLUFINE®Sulfate (Chisso Corporation, Osaka JAPAN) Column Purification; EluateDilution and Creation of Sub-Lots; Formulation with Alum, and BulkVaccine Drug Product Formulation.

Additional process development studies were initiated, with the goals ofreducing residual Vero HCP; increasing overall virus recovery; removingresidual sucrose in the virus sample; and minimizing aggregation of thepurified virus particles. The result of the development work was animproved downstream purification process, the overall schematic of whichis represented in in the flow chart on the right side of each of FIG. 6and FIG. 7, “Downstream New Process.”

In one embodiment of the invention, the New Downstream Process outlinedin the right side of FIG. 6 can have the following steps, eachrepresented by a rectangle in the Figure: Following Virus Harvest, (1)Clarification using a depth filter, and buffer adjustment. (2)BENZONASE® digestion and 0.2 Micron Filtration, producing a live virusbulk. (3) Purification using CELLUFINE® Sulfate Chromatography andDilution for inactivation step. (4) Virus Inactivation and 0.45 MicronFiltration. (5) Sucrose Gradient UltraCentrifugation. (6) IdentifyFractions; Pool Fractions; Warm to from about 25° C. to about 30° C.;0.2 Micron Filtration, forming Bulk Drug Substance, a purified,inactivated virus. (7) Alum Binding and Formulation.

In another embodiment of the invention, the New and Improved DownstreamProcess outlined in the right side of FIG. 7 can have the steps as inthe New Downstream Process shown in FIG. 6, with the following changes.Just prior to the virus inactivation step, there is a Purification Stepusing GE CAPTO™ Core 7FT with one percent (Human Serum Albumin) HSA orrHSA and adjustment to 20 percent sucrose/0.0005 percent TWEEN™-20. Thenfollowing the virus inactivation, there is an adjustment with onepercent human albumin or rhuman albumin and 0.2 micron filtration toyield the bulk drug substance which can then be formulated with Alum,buffer exchange, and stabilizers.

It should be noted that the virus data determined by the 2E10 monoclonalantibody is elevated when bound to alum. While not being bound bytheory, we postulated that the virus particle is arranged on the surfaceof the alum hydrogel so that the epitope may be presented in a more openform. Another hypothesis is that the particles are arranged in a moresymmetrical manner, thus precluding the formation of aggregates whichcould mask epitope exposure.

These experiments indicate that the recovery of intact virus may be insome way dependent upon the presence of a small amount of “chaperone”protein. We have discovered, unexpectedly, that increasing the postsucrose gradient purified pool temperature prior to filtration resultsin significantly greater recovery of virus prior to alum binding,thereby resulting in an increase in virus recovery. This effect oftemperature may be dependent upon the presence of the “chaperone”protein at a sufficient concentration.

Virus Harvest and BENZONASE® Treatment

The conditioned cell culture medium containing virus was removed fromthe bioreactor and clarified by depth filtration. A Millipore DE50 depthfilter was used to clarify the Virus Harvest. The depth filter wasflushed twice, once with USP purified H₂O followed by buffer with thetarget formulation 20 mM Tris, 145 mM NaCl, pH 8. The harvest materialwas passed through the depth filter at a flow rate of approximately 500mL/min and a pressure not to exceed 25 psi.

Filtered material was collected into a bioprocess single-use bag. Thedepth filter was chased with the same buffer and the chase volume wascombined with the original filtrate. Following depth filtration, thematerial was adjusted to a target formulation of 50 mM Tris, pH 8 and 2mM MgCl2 in preparation for the subsequent BENZONASE® treatment step.The adjusted clarified harvest was mixed for approximately 10 min atroom temperature.

The adjusted clarified virus intermediate was treated with BENZONASE® inorder to fragment Vero cell DNA. BENZONASE® was added to the adjustedclarified virus to a final target concentration of 3 units/mL, and thesuspension was mixed for 16 to 18 hours at room temperature. AfterBENZONASE® treatment the product pool was 0.5 μm filtered.

CELLUFINE® Sulfate

The virus was further purified and concentrated by CELLUFINE® sulfatechromatography. CELLUFINE® sulfate (Chisso, Tokyo, Japan) is a virusaffinity resin designed to concentrate, purify and depyrogenate virus.This process step significantly reduces Vero cell proteins, endotoxinand Vero cell DNA. The 2E10 ELISA that detects a YF viral envelopeepitope is used to measure the virus concentration on a sample of theclarified and BENZONASE®-treated live virus to ensure the column isappropriately sized to process a virus mass challenge of no more than5-6E+09 VE per/ml of CELLUFINE® sulfate resin.

Prior to loading the live virus material, the column was charged with0.1 M NaoH/0.5 M NaCl buffer at an approximate linear flow rate of 200cm/hr. The column was then equilibrated with equilibration buffer, 10 mMTris, 145 mM NaCl, pH 7.5 at an approximate linear flow rate of 200cm/hr. Post equilibration, the appropriate volume of the virus wasloaded onto the column at an approximate linear flow rate of 200 cm/hr.

After loading, the column was washed with 10 mM Tris, 145 mM NaCl, pH7.5 buffer at an approximate linear flow rate of 200 cm/hr. The boundvirus was eluted from the column using 10 mM Tris, 1.5 M NaCl, pH 7.5buffer at a reduced linear flow rate of approximately 100 cm/hr.Decreasing the elution flow rate increases buffer residence time and,therefore, decreases elution volume.

The elution pool was immediately diluted 2× (1 part eluate to 1 partdilution buffer) with 62.5 mM HEPES, pH 8 (target formulation) buffer toreduce precipitation of virus. Post elution, the resin was cleaned with0.1N NaOH/0.5 M NaCl at an approximate linear flow rate of 200 cm/hr.The column was stored in 0.1N NaOH/0.5 M NaCl at room temperature.

GE CAPTO™ Core 700

The 2× diluted CELLUFINE® Sulfate elution pool was then loaded onto aCAPTO™ Core 700 column. Prior to loading, the column was regeneratedwith 0.1N NaOH/0.5M NaCl at a linear flow rate of 300 cm/hr. One columnvolume of a re-equilibration buffer of 500 mM Tris/145 mM NaCl, pH 7.5was applied prior to the equilibration buffer consisting of 20 mMMES/100 mM NaCl, pH 7 at a linear flow rate of 300 cm/hr.

After loading, the column was washed with 20 mM MES/100 mM NaCl, pH 7 ata linear flow rate of 300 cm/hr. The flow through and wash was collectedas the product. This fraction was then diluted 2× with 50 mM HEPES/20%Sucrose/0.001% TWEEN™-20, pH 8. The resin was cleaned with 1N NaOH/1MNaCl at a linear flow rate of 300 cm/hr. The column was stored in 0.1NNaOH/0.5M NaCl at room temperature.

ß-PL Inactivation

A 10% solution of BPL was made by diluting BPL with water for injection(WFI). The 10% BPL was stored in single use aliquots at <−60° C. Theconcentration of BPL in this 10% solution was confirmed by gaschromatography (GC) analysis.

Human serum albumin (HSA) was added to the sample to adjust theconcentration to 1 mg/mL HSA. A sufficient amount of 10% BPL was thawedand added while mixing to the live virus pool to bring the BPLconcentration to approximately 0.1% (v/v) BPL.

The inactivation mixture was mixed for approximately 3 hours at roomtemperature on a low heat-generating stir plate. This material was thenincubated at 30° C. for 60 minutes, and filtered using a 0.2 m PESfilter.

Alum Binding and Formulation

The 0.2 μm filtered purified inactivated virus was bound to “alum”[Aluminum aluminum Hydroxide hydroxide (Alum ALHYDROGE®)] and bufferexchanged into the final formulation buffer. All process steps wereaseptically performed.

One part of 2% Alum alum was added to 9 parts of 0.2 μm filtered sucrosegradient-purified inactivated virus to achieve a final alum targetconcentration of 0.2%. We refer to the resulting product as the originalalum-bound virus pool. The pool was mixed for a target of 1-4 hours atroom temperature.

The alum-bound virus was aseptically buffer exchanged into 10 mMTris/1.2 mM MgCl2/10 mM L-glutamic acid/0.11 mM D-mannitol/2 mMTrimethylamine-N-oxide dihydrate, pH 7.5. The alum-bound virus wassettled by centrifugation or membrane filtration. After settling, thesupernatant was decanted. The alum-bound virus pellet was re-suspendedwith formulation buffer to a volume equal to the original alum-boundvirus pool volume and mixed for a target of 10 minutes at roomtemperature. This process was repeated 3-4× until the alum-bound viruspool was exchanged into the final formulation buffer.

The alum-bound and buffer exchanged virus pool was stored at 2-8° C.This is referred to herein as the “Bulk Drug Product.” The potency ofthe Bulk Drug Product was measured using ELISA that detects alum-boundYF.

Process recovery chromatograms for Experiments Y1626A, Y1632A, andY1637A are shown in FIG. 1 through FIG. 4, respectively.

A process recovery chromatogram for Experiment 1639A using a CELLUFINE®Sulfate Column is shown in FIG. 4 and for Experiment 1639A using aCAPTO™ DeVirS column in FIG. 5.

Two other embodiments of the disclosed process are shown in FIG. 8,“Downstream new and Improved process #3.”

It should be noted that the experiments were run using frozen/thawedmaterials and that there was no non-GMP material remaining. We recommendusing stored GMP material for further development of the disclosedprocess. However, the experimental results obtained with the non-GMPmaterial demonstrate that the disclosed process significantly reducesHCP and DNA levels.

The disclosed chromatographic purification process yielded sufficientvirus recovery and reduced the host cell protein levels to below thelimit of detection of the commercial Vero cell HCP assay. These virusrecoveries and HCP results were comparable to results achieved by asimilar purification process utilizing sucrose gradientultracentrifugation. A benefit of the disclosed process is that, unlikecentrifugation methods, it is suitable for use in single-use systems.

The next phase of the Yellow Fever purification development is toreplace the Chisso CELLUFINE® Sulfate resin with an equivalent GEHealthcare resin. The CELLUFINE® sulfate resin can potentially bereplaced with GE Healthcare's CAPTO™ DeVirS resin. The CAPTO™ DeVirS ispart of GE Healthcare's Custom Designed Media program, and is anaffinity chromatography resin with the ligand dextran sulfate, which isknown to have an affinity-like behavior to different types of virus.CAPTO™ DeVirS offers the a number of benefits for purification of virus,including, for example, excellent productivity, good chemical stability,and affinity-like behavior to various viruses.

The next planned experiments include following the use of CAPTO™ DeVirSwith GE CAPTO™ Core 700. We will also investigate the use of an anionexchange membrane such as a Q Monolith (BIA) or a Q membrane (Natrix,Pall, Sartorius) between the step using the CAPTO™ DeVirS and the stepusing GE CAPTO™ Core 700.

EQUIVALENTS

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith. All of the features disclosed in this specification(including any accompanying claims, abstract and drawings), and/or allof the steps of any method or process so disclosed, may be combined inany combination, except combinations where at least some of suchfeatures and/or steps are mutually exclusive. The invention is notrestricted to the details of any foregoing embodiments. The inventionextends to any novel one, or any novel combination, of the featuresdisclosed in this specification (including any accompanying claims,abstract and drawings), or to any novel one, or any novel combination,of the steps of any method or process so disclosed.

What is claimed is:
 1. An essentially pure inactivated flaviviruspreparation made by the process of: a. obtaining a composition suitablefor purification from a virus-infected Vero cell culture; b. clarifyingthe composition obtained in step “a” by removing cells and cell debrisfrom said composition by depth filtration; c. subjecting the filtrate instep “b” to endo-nuclease digestion and 0.2 Micron Filtration sufficientto fragment Vero cell DNA followed by filtration; d. purifying thefiltrate obtained in step “c” via a cross-linked agarose matrix with adextran sulfate functional group chromatography resin and a cross-linkedagarose matrix with an octylamine ligand chromatography resin having ashell that excludes molecules with a molecular mass greater thanapproximately 700 kDa from passing through pores of the shell to achievedepyrogenation of said virus in said filtrate; e. stabilizing andinactivating the viral product obtained in step “d” with an inactivatingamount of ß-PL, and then performing sucrose gradientultra-centrifugation on the viral product; f. after inactivating theviral product in step “e”, adjusting the temperature of the viralproduct to above about 25° C. to about 30° C.; g. filtering the productobtained in step “f”; and h. formulating the product obtained in step“g” bound to alum.